The proteasome is a key enzyme in the ubiquitin-dependent pathway of degradation. It is important in human health and is responsible for proper regulation of a wide variety of basic cell functions. Historically, little attention has been given to how the proteasome is regulated. Our recent studies in yeast have identified unanticipated mechanisms of proteasome regulation. The two mechanisms involve a proteasome-associated deubiquitinating enzyme, Ubp6, whose mammalian ortholog is Usp14: (i) Ubp6 inhibits the activity of the proteasome, and (ii) the expression of Ubp6, and in turn the amount of Ubp6 on the proteasome, are negatively regulated by ubiquitin levels. These two mechanisms are likely related, in that low ubiquitin levels induce Ubp6, which then inhibits proteasome activity, which has the effect of reducing the destruction of scarce ubiquitin. Our preliminary data indicate that, as predicted from our findings in yeast, Usp14 inhibits mammalian proteasomes. In Aim 1, we will characterize the mechanism of this inhibition in mammals and its effects on cell function, using purified proteasomes and purified Usp14 as well as an existing line of usp14 null MEF cells. In Aim 2, we will attempt to isolate the molecular machinery of the ubiquitin stress response. We present preliminary data that the ubiquitin stress response, which we first identified in yeast, may operate in mammals as well. We will use both yeast and mammalian systems to dissect the response. A large number of diseases involve protein misfolding and its pathological effects. It is well appreciated that misfolded proteins are preferred substrates for the ubiquitin-proteasome pathway. Thus, in many disease states, misfolded proteins may impose an unusually high load on the ubiquitin-proteasome system. In such instances, it may be necessary for the system to maintain function through compensatory regulatory mechanisms. Therefore, the types of regulatory mechanisms that will be probed here for the first time in mammals may potentially be relevant to the pathogenesis of multiple diseases. PUBLIC HEALTH RELEVANCE The proteasome is a key enzyme that controls the levels of hundreds of proteins and thus is responsible for the proper regulation of a wide variety of basic cell functions. Our recent studies in yeast have identified unanticipated mechanisms by which this enzyme is itself controlled. The grant is designed mainly to test whether these interesting new mechanisms may apply to mammals. If so, the work may advance our understanding of human health, because the ubiquitin-proteasome system plays important roles in both cancer and neurodegenerative disease.